During the last 30 years, two main categories of nanometer-scale systems have been developed for use as carriers for molecules of interest: polymer systems and lipid systems.
The first of these were shown to be relatively disappointing in terms of their industrial application, likely for reasons of toxicity. Furthermore, the formulations available on the market are predominantly based on lipids that have generated two large families of carriers: liposomes and lipid particles (nanoemulsions (NE), nanostructured lipid carriers (NLC), solid lipid nanoparticles (SLN)). Liposomes, and to a lesser extent nanoemulsions, have given rise to numerous cosmetic applications and several medicines on the market, while nanostructured or solid lipid particles, developed more recently, are present in many cosmetic products and in clinical trials for the pharmaceutical sector.
A liposome is defined as an artificial structure consisting of one or more concentric lipid bilayers, confining between them compartments of water or aqueous buffer. Liposomes are prepared from a single type, or from several types, of natural or synthetic phospholipids organized such that the polar heads come together so as to create the bilayer. The most traditional method of preparing liposomes is so-called lipid film hydration. Liposomes are increasingly being developed as carriers for hydrosoluble, liposoluble and amphiphilic active principles. The encapsulation of active principles in the aqueous phase or the lipid bilayer thus makes it possible to protect said principles from enzymatic degradation or elimination by the immune system, but also to decrease their possible toxic side effects (e.g., hemolysis, thrombophlebitis, blood coagulation) when administered parenterally. (Meure et al., Aaps Pharmscitech, 9:798-809, 2008; Storm and Crommelin, Pharmaceutical Science & Technology Today, 1:19-31, 1998) [1, 2]. At the root of a dozen commercial compositions (e.g., Myocet®, Doxil®/Caelyx®, AmBisome®, Visudyne®, etc.), liposomes have several major disadvantages, however: they lack specificity for the target cell, the oxidation and physical instability of phospholipids requires them to be lyophilized, they are delicate to produce industrially, and there is a certain limit to the amount of molecules of interest that can be encapsulated. Indeed, amphiphilic or lipophilic molecules are able to combine with liposomes by insertion into their membranes, but at the risk of destabilization of the latter.
Emulsions are fine dispersions of droplets of one liquid (dispersed phase) in another (dispersant or continuous phase), the two liquids being relatively immiscible; they are most often of the water/oil type. The term “nanoemulsion” (NE) is used when the particle size obtained is very small, i.e., a mean size of about a hundred nanometers. They are generally produced by mechanical fragmentation of an oil phase in an aqueous phase, and optionally stabilized by the presence of surfactant. Compared to conventional emulsions, the small size of the globules gives them advantageous pharmaceutical properties, in particular in terms of physical stability during storage and possible routes of administration, in particular intravenous administration requiring the use of small droplets. However, these systems can incorporate only very lipophilic active principles that are soluble in the component oils of these emulsions (soybean oil, olive oil), thus limiting their potential applications.
Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) were developed to increase the physicochemical stability of encapsulated active principles and the post-administration stability of lipid carriers as a whole, generally for eventual use in cosmetics, by virtue of their properties of adhesion, occlusion and skin hydration, and in pharmaceuticals for administering and protecting active principles of interest. (Bunjes, Current Opinion in Colloid & Interface Science, 16(5):405-411, 2011; Harde et al., Expert Opinion on Drug Delivery, 8(11):1407-1424, 2011; Harms et al., Journal of Drug Delivery Science and Technology, 21(1):89-99, 2011; Joshi and Muller, European Journal of Pharmaceutics and Biopharmaceutics, 71:161-172, 2009; Muller et al., Current Drug Discovery technologies, 8(3):207-227, 2011; Pardeike et al., International Journal of Pharmaceutics, 366:170-184, 2009; Souto and Doktorovova, Methods in Enzymology, 464:105-129, 2009) [3-9]. As with the nanoemulsions described above, the very high lipophilicity of the raw materials used limits the choice of potentially administrable active principles. Moreover, it was shown that the polymorphism of lipids in the solid state has a large influence on the physical stability of these systems (expulsion of active molecules, gelation), in particular in the case of SLN.
Lipid nanocapsules developed by the University of Angers were obtained by a phase-inversion method and are surrounded by a phospholipid monolayer. Although very similar to SLN and NLC type nanodispersions, these particles have been described as nanocapsules stabilized by a crystallized layer of phospholipids and a nonionic polyoxyethylene surfactant (international application WO 01/64328; Huynh et al., Journal of Pharmaceutics, 379:201-209, 2009) [10, 11]. Advantageous because it is relatively “mild,” the phase-inversion technique requires the use of relatively specific raw materials and very fine control of temperatures during the preparation process. These aspects can limit the large-scale development of this approach.
A few years ago, submicron-scale cationic emulsions consisting of two-compartment oil/water structures, called “handbags,” were developed (FIG. 1). In order to use them as carriers for active principles, their formation requires the presence of stearylamine, which supports the mass insertion of triglycerides into the lipid bilayer delimiting the aqueous compartment (Texeira et al., Pharmaceutical Research, 17:1329-1332, 2000) [12]. Unfortunately, the proportion of these two-compartment objects remains in the minority (less than 20%) among a multitude of other objects formed during the preparation process (micelles, liposomes, nanoemulsions). Moreover, the use of a cationic surfactant like stearylamine should be considered with caution considering the toxicity of this type of product to negatively-charged biological membranes.
More marginal, nanoemulsions of particles having a diameter of 10-250 nm (called emulsomes or ultrasomes) comprising a lipid core consisting of a lipid in liquid or solid form surrounded and stabilized by at least one phospholipid bilayer, as in liposomes, were developed and designed for the parenteral, oral, rectal, intranasal or topical administration of liposoluble or hydrosoluble molecules (U.S. Pat. No. 5,576,016; Gupta et al., Journal of Drug Targeting, 15:437-444, 2007; Gupta and Vyas, Journal of Drug Targeting, 15:206-217, 2007; Kretschmar et al., Mycoses, 44:281-286, 2001; Paliwal et al., International Journal of Pharmaceutics, 380:181-188, 2009; Wu et al., Journal of Immunology, 185(6):3401-3407, 2010) [13-18]. The method for preparing these emulsomes is applied with difficulty on an industrial scale because it generally requires the use of an organic solvent and the deposition and rehydration of a phospholipid film. Indeed, these particles are mainly obtained by a phospholipid film hydration technique very similar to that employed for liposomes, except that the aqueous phase contains preformed lipid nanoparticles. The final particle results from the “statistical confinement” of oil droplets in the phospholipid bilayers. Consequently, the process generates a priori various populations of objects (emulsomes, liposomes, nanoemulsions or solid nanoparticles) with no uniting of the lipid and phospholipid parts. This can a priori cause system stability problems during certain purification operations (centrifugation, for example) or during storage.
There is thus a genuine need for lipid carriers that sweep aside these defects, disadvantages and obstacles of the prior art, in particular for a simple production method making it possible to control the long-term stability of lipid carriers for administering a large amount of molecules of interest having a wide range of polarity and, optionally, to envisage the co-encapsulation of hydrophilic and lipophilic active principles in the same nano-object